Cardiovascular disease is a critical health issue in Western countries. The leading cause of death is heart failure (HF), which represents not only a significant problem to be addressed but also involves a large amount of health care costs. According to the American Heart Association, over 5.5 million patients were diagnosed with congestive HF in the U.S. The estimated annual direct and indirect healthcare costs associated with chronic HF in the U.S. alone exceeds $24 billion. The projection of morbidity and mortality will be continuously increased in the next 15 years because of the significant increase in population. Therefore, improvement of HF therapy is extremely important.
β-blockers are now widely used for treating HF; however, despite the well established effect in clinical trials, some patients are intolerant to β-blocker therapy because they occasionally exacerbate HF by attenuation of cardiac contraction (Bristow M R. Circulation. 2000; 101(5):558-569; Hunt et al. Journal of the American College of Cardiology. 2005; 46(6):e1-e82; Ko et al. Arch Intern Med. 2004; 164(13):1389-1394.). Therefore, a new approach to β-blocker therapy is indicated and could save such patients.
HF is the common endpoint of many different forms of heart disease, and a pathophysiologic state with impaired cardiac function such that the heart cannot provide a sufficient output for organs and tissues. Despite that, developments in medical treatments have resulted in reducing the overall mortality rate from heart disease over the last several decades; however, death from chronic HF still continues to increase. With chronic HF, sympathetic activity is known to be increased to compensate for impaired cardiac function. Such increase of sympathetic activity stimulates cardiac contractility, thus, HF is improved. However, paradoxically, elevated sympathetic activity also causes myocardial apoptosis. Myocardial apoptosis results in a loss of cardiac myocytes, thus, contractile function is impaired. Increased oxidative stress is also a major causal factor for the progression of HF (Giordano et al. J Clin Invest. March 2005; 115(3):500-508.).
AC is a 12-transmembrane protein that catalyzes the conversion of ATP to cAMP upon the stimulation of various G-protein coupled receptors such as β-adrenergic receptor (β-AR). Nine mammalian AC subtypes have been identified, and each subtype shows distinct tissue distributions, and biological and pharmacological properties (Iwatsubo et al., Endocr Metab Immune Disord Drug Targets. September 2006; 6(3):239-247). Stimulation of G protein-coupled receptors induces binding of the stimulatory Gα subunit (Gsα) to AC, and enhances its catalytic activity to convert ATP into cAMP. cAMP regulates multiple downstream molecules, via protein kinase A (PKA) and exchange protein activated by cAMP (Epac).
A series of studies in genetically-engineered mice has demonstrated the crucial role of AC5, a major cardiac subtype of AC, in progression of heart failure (HF). Disruption of AC5 protects against the development of several type of HF (Okumura et al., Circulation. Oct. 16, 2007; 116(16):1776-1783; Okumura et al., PNAS. Aug. 19, 2003; 100(17):9986-9990; Yan et al., Cell. Jul. 27, 2007; 130(2):247-258). Interestingly, prevention of aging-related HF resulted in prolonged lifespan; therefore, the development of a chemical inhibitor of AC5 would be extremely valuable.
AC5 is a major cardiac subtype of AC, which provides 20% of total AC activity in the heart, and recent studies including ours revealed its crucial role in progression of HF (Iwatsubo et al., J Biol. Chem. Sep. 24, 2004; 279(39):40938-40945; Okumura et al., Circ Res. Aug. 22, 2003; 93(4):364-371). AC5KO mice showed decreased myocardial apoptosis and preserved cardiac function in HF models induced by chronic pressure overload (Okumura et al. Proceedings of the National Academy of Sciences. 2003; 100(17):9986-9990), chronic β-AR stimulation (Okumura et al., Circulation. 2007; 116(16):1776-1783) and aging (Yan et al., Cell. Jul. 27, 2007; 130(2):247-258). In all these HF models, myocardial apoptosis, which is a major cause for progression of HF, was significantly decreased in AC5KO, indicating that AC5 plays a central role in inducing apoptosis and subsequent development of HF. Moreover, AC5Tg showed decreased left ventricular ejection fraction (LVEF) and increased apoptosis in response to chronic pressure overload, indicating that AC5 accelerates the progression of HF by inducing myocardial apoptosis. These data strongly suggest that among mechanisms by which myocardial apoptosis occurs such as renin-angiotensin-aldosterone, death receptor and calcium signaling, sympathetic activity overdrive, particularly via stimulating AC5, plays a major role in inducing myocardial apoptosis and development of HF.
Classic inhibitors of AC, known as P-site inhibitors, have been studied since the 1970's. It was first thought that there was an adenosine-reactive site within intracellular domain of AC, the “P” site, which inhibits the catalytic activity of AC. In spite of their similar chemical structure to the substrate ATP, P-site inhibitors showed un- or non-competitive inhibition with respect to ATP, indicating little influence on molecules which have ATP-binding site (Londos et al., Proc Natl Acad Sci USA. December 1977; 74(12):5482-5486). Although it has been a very attractive idea to develop P-site inhibitors with enhanced AC subtype selectivity, few attempts have been successful due to the difficulties of experiments in which the selectivity of each AC isoforms can be examined in vitro. However, several groups including ours have developed such experimental systems using the baculovirus-based recombinant AC overexpression system (Iwatsubo et al., J Biol. Chem. Sep. 24, 2004; 279(39):40938-40945; Onda et al. J Biol. Chem. Dec. 21, 2001; 276(51):47785-47793).
9-β-D-arabinofuranosyladenine (Ara-Ade) contains an adenosine-like structure where the adenine ring is essential not only for binding to the AC catalytic core but also for penetrating the plasma membrane (Iwatsubo et al. J Biol. Chem. 2004; 279(39):40938-40945, Onda et al. J Biol. Chem. 2001; 276(51):47785-47793. Tesmer et al. Biochemistry. 2000; 39(47):14464-14471. Tesmer et al. Science. 1999; 285(5428):756-760). For example, NKY80, which does not contain adenosine within its structure, showed moderate inhibition of purified AC5 protein in vitro, but it did not inhibit cAMP accumulation in cultured cardiac myocytes, indicating that the adenosine structure seems essential for penetrating the plasma membrane (Iwatsubo, et al. J Biol. Chem. 279(39):40938-40945). In addition, adenosine hardly crosses through the blood-brain barrier (BBB) (Isakovic et al. Journal of Neurochemistry. 90(2):272-286.), having little influence on brain function; this is important because AC5 is also expressed in the striatum other than the heart, thus by passing BBB AC5 inhibitors may cause adverse effects in the brain.